Mario was raised in Seattle, Washington by Marilyn Batali and Armando Batali,[6] who worked for Boeing most of Mario’s childhood. He spent his high school years studying in Madrid, Spain before attending Rutgers University for Spanish Theater and Business Management. During his college years he worked for a stromboli restaurant and bar near the Rutgers campus called Stuff Yer Face in New Brunswick, New Jersey. After graduating, he attended Le Cordon Bleu in London for a few short months before taking an apprenticeship with revered Chef Marco Pierre White in Borgo Capanne, a small northern Italian village. Over the next three years he underwent a culinary transformation that prepared him to leap into New York’s Italian cuisine with his own unique signature.

Currently, Mario is living in Greenwich Village with his wife Susi Cahn, of Coach Dairy Goat Farm, and his two sons Leo and Benno. He keeps a summer home on Grand Traverse Bay in tiny Northport, Michigan.

Mario teamed up with Joe Bastianich to form B&B Hospitality Group. Together they opened Babbo Ristorante e Enoteca in New York City which quickly gained the coveted three stars award from the New York Times, the first Italian restaurant to do so in 40 years. Together they opened seven more restaurants in New York: Lupa, Esca, Casa Mono, Bar Jamon, Otto, Del Posto, and Eataly (an Italian marketplace). In 2010, Del Posto received a four-star review from the New York Times. It is one of only six restaurants in New York with the award. Their culinary empire has expanded to 10 restaurants in New York, four restaurants in Las Vegas, two restaurants in Los Angeles, two restaurants in Singapore, one Italian market in Chicago, and two restaurants in Hong Kong. In 2015 they opened Babbo Pizzeria e Enoteca in the Seaport area of Boston

Batali is a critic of hydraulic fracturing, commonly known as fracking, a method of natural gas extraction. He has signed onto the cause of Chefs for the Marcellus, whose mission is to "protect [New York's] regional foodshed from the dangers of hydraulic fracturing for natural gas (fracking)."[9][10] In May 2013, Batali co-wrote an opinion article with chef Bill Telepan for the New York Daily News, in which the two wrote that "Fracking ... could do serious damage to [New York's] agricultural industry and hurt businesses, like ours, that rely on safe, healthy, locally sourced foods."[11]

The Mario Batali Foundation was founded in 2008 to ensure that all children are well read, well fed, and well cared for.

Mario aids the Lunchbox Fund which was founded as a nonprofit to ensure that orphaned and vulnerable school children in the rural areas of South Africa are given at least one meal a day.

He is also involved with the Food Bank for New York City which was founded to provide meals and support to the most vulnerable in New York City through soup kitchens, food pantries, classrooms, afternoon programs, and more.